The mu opioid receptor (MOR) plays an important role in mediating analgesia, euphoria, and addiction through opiates such as morphine. Although the behavioral effects of opiates have been characterized at length, much remains unknown with regards to the cellular mechanisms by which morphine regulates MORs and how they contribute to the physiological effects of opiates. MORs are co-expressed with neurokinin 1 (NK1) receptors in several regions of the central nervous system (CNS), including a subset of neurons in the striatum, a brain region important for reward processing relevant to drug addiction. Although these two neurochemical systems have been shown to have an interaction at the behavioral level, what remains unknown is whether there is cross-regulation between these receptor types at the cellular level. In preliminary experiments, I examined simultaneous activation of epitope-tagged MORs and NK1 receptors in rat striatal neurons and neurbblastoma 2A cells. While morphine normally promotes MOR endocytosis in these cell types, quantificiation by immunofluorescence microscopy indicated that activation of NK1 receptors with substance P greatly inhibited the ability of morphine to drive MOR endocytosis. This effect seems to be related to cytoplasmic levels of the regulatory protein p-arrestin. In my first specific aim, I will further examine this cross-regulation of MORs on a biochemical level using surface biotinylation. I will also investigate the cellular mechanism underlying NK1 receptors' inhibitory effect on MOR endocytosis by mutations of the NK1 receptor that are known to disrupt its association with p-arrestin. My second aim will focus on examining the signaling consequences of this MOR-NK1 receptor interaction. In particular, I will investigate whether inhibiting MOR endocytosis affects its acute desensitization and resensitization in response to morphine. To begin to look at the physiological consequence of this receptor interaction, I will examine whether NK1 receptors' inhibitory effects can be observed with endogenous MORs and NK1 receptors in striatal neurons in my third aim. The goals of the present proposal are to better understand how other receptors and regulatory proteins occupying the same cellular environment may affect regulated MOR function. The proposed experiments will hopefully provide insight into how MORs may be functioning in important brain areas for reward and addiction. Morphine is one of the most effective analgesic, yet also highly addictive, drugs available. Understanding the cellular basis of opiate actions is important for furthering our knowledge of the mechanisms behind the actions of morphine. Ultimately, this will help lead to more effective treatment of public health issues relevant to pain and addiction. [unreadable] [unreadable] [unreadable]